Nuclear roles for non-lamin intermediate filament proteins.

The nuclear-localized lamins have long been thought to be the only intermediate filaments (IFs) with an impact on the architecture, properties, and functions of the nucleus. Recent studies, however, uncovered significant roles for IFs other than lamins (here referred to as "non-lamin IFs") in regulating key properties of the nucleus in various cell types and biological settings. In the cytoplasm, IFs often occur in the perinuclear space where they contribute to local stiffness and impact the shape and/or the integrity of the nucleus, particularly in cells under stress. In addition, selective non-lamin IF proteins can occur inside the nucleus where they partake in fundamental processes including nuclear architecture and chromatin organization, regulation of gene expression, cell cycle progression, and the repair of DNA damage. This text reviews the evidence supporting a role for non-lamin IF proteins in regulating various properties of the nucleus and highlights opportunities for further study.

A role for keratin 17 during DNA damage response and tumor initiation.

High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including γ-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas.

Bone Mineral Density: Clinical Relevance and Quantitative Assessment.

Bone mineral density (BMD) measurement by dual-energy x-ray absorptiometry (DXA) is an internationally accepted standard-of-care screening tool used to assess fragility-fracture risk. Society guidelines have recommended which populations may benefit from DXA screening and the use of the fracture risk assessment tool (FRAX) to guide decisions regarding pharmacologic treatment for osteoporosis. According to the U.S. National Osteoporosis Foundation guidelines, postmenopausal women and men at least 50 y old with osteopenic BMD warrant pharmacologic treatment if they have a FRAX-calculated 10-y probability of at least 3% for hip fracture or at least 20% for major osteoporotic fracture. Patients with osteoporosis defined by a clinical event, namely a fragility fracture, or with an osteoporotic BMD should also be treated. Patients who are treated for osteoporosis should be monitored regularly to track expected gains in BMD by serial DXA scans. With some drug therapies, BMD targets can be reached whereby further improvements in BMD are not associated with further reductions in fracture risk. Although reaching this target might suggest a stopping point for therapy, the reversibility of most treatments for osteoporosis, except for the bisphosphonates, has dampened enthusiasm for this approach. In the case of denosumab, it is now apparent that stopping therapy at any point can lead to an increase in multiple-fracture risk. For patients who do not respond to antiosteoporosis pharmacologic therapy with an improvement in BMD, or who have an incident fragility fracture on therapy, secondary causes of osteoporosis or non-compliance with medical therapy should be considered.

Keratin 17 regulates nuclear morphology and chromatin organization.

Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17-null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2ß (an isoform encoded by TMPO) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.This article has an associated First Person interview with the first author of the paper.

Types I and II Keratin Intermediate Filaments.

Keratins-types I and II-are the intermediate-filament-forming proteins expressed in epithelial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type-, differentiation-, and context-dependent manner. Here, we review how keratins serve multiple homeostatic and stress-triggered mechanical and nonmechanical functions, including maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications and keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility or altered tissue homeostasis. Furthermore, keratin mutation or misregulation represents risk factors or genetic modifiers for several additional acute and chronic diseases.

Keratins Are Going Nuclear.

Previously thought to reside exclusively in the cytoplasm, the cytoskeletal protein keratin 17 (K17) has been recently identified inside the nucleus of tumor epithelial cells with a direct impact on cell proliferation and gene expression. We comment on fundamental questions raised by this new finding and the associated significance.

Case diagnosis and characterization of suspected paralytic shellfish poisoning in Alaska.

Clinical cases of paralytic shellfish poisoning (PSP) are common in Alaska, and result from human consumption of shellfish contaminated with saxitoxin (STX) and its analogues. Diagnosis of PSP is presumptive and based on recent ingestion of shellfish and presence of manifestations consistent with symptoms of PSP; diagnosis is confirmed by detection of paralytic shellfish toxins in a clinical specimen or food sample. A clinical diagnostic analytical method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to evaluate the diagnosis of saxitoxin-induced PSP (STX-PSP) in 11 Alaskan patients using urine specimens collected between June 2010 and November 2011. Concentrations of urinary STX were corrected for creatinine concentrations to account for dilution or concentration of urine from water intake or restriction, respectively. Of the 11 patients with suspected PSP, four patients were confirmed to have STX-PSP by urine testing (24-364ng STX/g creatinine). Five patients had clinical manifestations of PSP though no STX was detected in their urine. Two patients were ruled out for STX-PSP based on non-detected urinary STX and the absence of clinical findings. Results revealed that dysphagia and dysarthria may be stronger indicators of PSP than paresthesia and nausea, which are commonly used to clinically diagnose patients with PSP. PSP can also occur from exposure to a number of STX congeners, such as gonyautoxins, however their presence in urine was not assessed in this investigation. In addition, meal remnants obtained from six presumptive PSP cases were analyzed using the Association of Official Analytical Chemists' mouse bioassay. All six samples tested positive for PSP toxins. In the future, the clinical diagnostic method can be used in conjunction with the mouse bioassay or HPLC-MS/MS to assess the extent of STX-PSP in Alaska where it has been suggested that PSP is underreported.

Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes.

Expression of the intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and in many tumors originating in stratified and pseudostratified epithelia. We report that autoimmune regulator (Aire), a transcriptional regulator, is inducibly expressed in human and mouse tumor keratinocytes in a K17-dependent manner and is required for timely onset of Gli2-induced skin tumorigenesis in mice. The induction of Aire mRNA in keratinocytes depends on a functional interaction between K17 and the heterogeneous nuclear ribonucleoprotein hnRNP K. Further, K17 colocalizes with Aire protein in the nucleus of tumor-prone keratinocytes, and each factor is bound to a specific promoter region featuring an NF-κB consensus sequence in a relevant subset of K17- and Aire-dependent proinflammatory genes. These findings provide radically new insight into keratin intermediate filament and Aire function, along with a molecular basis for the K17-dependent amplification of inflammatory and immune responses in diseased epithelia.

A high-throughput diagnostic method for measuring human exposure to organophosphorus nerve agents.

An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 µg/mL to 10.6 µg/mL, with an average concentration of 6.4 µg/mL.